Novel erythromelalgia treatment

ABSTRACT

The present invention is directed to the treatment of erythromelalgia comprising administering to a subject in need thereof, a therapeutically effective amount of 5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-prolinamide or a pharmaceutically acceptable salt, solvate or prodrug thereof.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/516,031, filed Mar. 31, 2017, which is a U.S. National Stage ofInternational Patent Application No. PCT/GB2015/052885, filed Oct. 2,2015, which claims the benefit of U.K. Patent Application No. 1417497.3,filed Oct. 3, 2014. Each of these applications is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to the treatment of erythromelalgiacomprising administering to a subject in need thereof, a therapeuticallyeffective amount of5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof.

BACKGROUND

Erythromelalgia is a disorder that typically affects the skin of thefeet or hands, or both, and causes visible redness, intense heat andsevere pain. In many cases the use of measures, such as the avoidance ofwarm temperatures or ongoing elevation required by some with severeerythromelalgia, mean that patients often have to make major adjustmentsto their lifestyles to avoid flare-ups. The cause of erythromelalgia isunknown in the vast majority of cases, and although a number ofmedications have been used to help relieve symptoms, there is no singlecure available. Erythromelalgia is a rare disease and approximately 15%of erythromelalgia cases have been associated with Nav1.7 gain offunction mutations (Dib-Hajj et al (2013) Nature Reviews Neuroscience14, 49-62; www.ghr.nlm.nih.gov/condition/erythrmelalgia).

Thus, there is a need to provide an effective treatment forerythromelalgia to help patients treat their symptoms.

WO 2007/042239 describes5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-prolinamide having thefollowing formula:

and pharmaceutically acceptable salts, solvates or prodrugs thereof.

Themistocleous et al. (2014) Pract. Neurol. 0:1-12 describes that gainof function mutations in Na_(v)1.7 are believed to cause erythromelalgiaand paroxysmal extreme pain disorder.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Whole cell patch-clamp electrophysiology analysis of thecompound of Example 2 on human recombinant WT (TTX-R version, Y362S) and4 mutated Nav1.7 channels from erythromelalgia patients (V400M, I234T,F1449V and S241T), stably expressed in HEK293 cells.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention, there is provided(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof for use inthe treatment of erythromelalgia.

According to a further aspect of the invention, there is provided amethod for the treatment of erythromelalgia comprising administering toa subject in need thereof, a therapeutically effective amount of(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof.

The invention also provides the use of(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide, or apharmaceutically acceptable salt, solvate or prodrug thereof, in themanufacture of a medicament for the treatment of erythromelalgia.

In one embodiment, the subject to be treated is a human.

A reference to(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide alsoincludes ionic forms, salts, solvates, isomers (including geometric andstereochemical isomers), tautomers, N-oxides, esters, prodrugs, isotopesand protected forms thereof; preferably, the salts or tautomers orisomers or N-oxides or solvates thereof; and more preferably, the saltsor tautomers or N-oxides or solvates thereof, even more preferably thesalts or tautomers or solvates thereof.

(5R)-5-(4-{[(2-Fluorophenyl)methyl]oxy}phenyl)-L-prolinamide can existin the form of a salt, for example an acid addition salt or, in certaincases a salt of organic and inorganic bases such as a carboxylate,sulfonate and phosphate salt. All such salts are within the scope ofthis invention, and references to(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide include thesalt forms of this compound.

The pharmaceutically acceptable salts of(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide are, forexample, non-toxic acid addition salts formed with inorganic acids suchas hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid,with carboxylic acids or with organo-sulfonic acids. Examples includethe HCl, HBr, HI, sulfate or bisulfate, nitrate, phosphate or hydrogenphosphate, acetate, benzoate, succinate, saccharate, fumarate, maleate,lactate, citrate, tartrate, gluconate, camsylate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate salts.For reviews on suitable pharmaceutical salts see Berge et al, J. Pharm,Sci., 66, 1-19, 1977; P L Gould, Int. J. Pharma., 33 (1986), 201-217;and Bighley et al, Encyclopedia of Pharmaceutical Technology, MarcelDekker Inc, New York 1996, Volume 13, page 453-497.

The salts described herein can be synthesized from the parent compoundthat contains a basic or acidic moiety by conventional chemical methodssuch as methods described in Pharmaceutical Salts: Properties,Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth(Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002.Generally, such salts can be prepared by reacting the free acid or baseforms of these compounds with the appropriate base or acid in water orin an organic solvent, or in a mixture of the two; generally,non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, oracetonitrile are used.

Acid addition salts (mono- or di-salts) may be formed with a widevariety of acids, both inorganic and organic. Examples of acid additionsalts include mono- or di-salts formed with an acid selected from thegroup consisting of acetic, 2,2-dichloroacetic, adipic, alginic,ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic,4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic,(+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic,citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric,gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic),glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric,hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic),isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic,maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic,naphthalene-2-sulfonic, naphthalene-1,5-disulfonic,1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic,palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic,salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric,tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic andvaleric acids, as well as acylated amino acids and cation exchangeresins.

One particular group of salts consists of salts formed from acetic,hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic,succinic, maleic, malic, isethionic, fumaric, benzenesulfonic,toluenesulfonic, methanesulfonic (mesylate), ethanesulfonic,naphthalenesulfonic, valeric, acetic, propanoic, butanoic, malonic,glucuronic and lactobionic acids. One particular salt is thehydrochloride salt.

The compounds of the invention may exist as mono- or di-salts dependingupon the pK_(a) of the acid from which the salt is formed.

It will be appreciated that for use in medicine the salts of thecompounds should be pharmaceutically acceptable. Suitablepharmaceutically acceptable salts will be apparent to those skilled inthe art. Pharmaceutically acceptable salts include those described byBerge, Bighley and Monkhouse, J. Pharm. Sci. (1977) 66, pp. 1-19. Suchpharmaceutically acceptable salts include acid addition salts formedwith inorganic acids e.g. hydrochloric, hydrobromic, sulphuric, nitricor phosphoric acid and organic acids e.g. succinic, maleic, acetic,fumaric, citric, tartaric, benzoic, p-toluenesulfonic, methanesulfonicor naphthalenesulfonic acid. Other salts e.g. oxalates or formates, maybe used, for example in the isolation of compounds described herein, andare included within the scope of this invention. However, salts that arenot pharmaceutically acceptable may also be prepared as intermediateforms which may then be converted into pharmaceutically acceptablesalts. Such non-pharmaceutically acceptable salts forms, which may beuseful, for example, in the purification or separation of the compoundsof the invention, also form part of the invention.

In one embodiment, the(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof is selectedfrom (5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamidehydrochloride or(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamidemethanesulfonate. In a further embodiment, the(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof is(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamidehydrochloride.

Certain of the compounds described herein may form acid addition saltswith one or more equivalents of the acid. The present invention includeswithin its scope all possible stoichiometric and non-stoichiometricforms.

Those skilled in the art of organic chemistry will appreciate that manyorganic compounds can form complexes with solvents in which they arereacted or from which they are precipitated or crystallized. Thesecomplexes are known as “solvates”. For example, a complex with water isknown as a “hydrate”. Pharmaceutically acceptable solvates of thecompound of the invention are within the scope of the invention. In oneembodiment, the pharmaceutically acceptable solvates of the compounds ofthe invention include the hydrate thereof.

It will be appreciated by those skilled in the art that certainprotected derivatives of(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide, which maybe made prior to a final deprotection stage, may not possesspharmacological activity as such, but may, in certain instances, beadministered orally or parenterally and thereafter metabolised in thebody to form compounds of the invention which are pharmacologicallyactive. Such derivatives may therefore be described as “prodrugs”. Allsuch prodrugs of compounds of the invention are included within thescope of the invention. Examples of suitable pro-drugs for the compoundsof the present invention are described in Drugs of Today, Volume 19,Number 9, 1983, pp 499-538 and in Topics in Chemistry, Chapter 31, pp306-316 and in “Design of Prodrugs” by H. Bundgaard, Elsevier, 1985,Chapter 1 (the disclosures in which documents are incorporated herein byreference). It will further be appreciated by those skilled in the art,that certain moieties, known to those skilled in the art as“pro-moieties”, for example as described by H. Bundgaard in “Design ofProdrugs” (the disclosure in which document is incorporated herein byreference) may be placed on appropriate functionalities when suchfunctionalities are present within compounds of the invention.

Those skilled in the art of organic chemistry will appreciate that manyorganic compounds can form complexes with solvents in which they arereacted or from which they are precipitated or crystallized. Thesecomplexes are known as “solvates”. For example, a complex with water isknown as a “hydrate”. Pharmaceutically acceptable solvates of thecompound of the invention are within the scope of the invention.

As used herein, unless otherwise noted, the term “erythromelalgia” (alsoknown as Gerhardt disease, Mitchell disease or Weir-Mitchell disease)shall mean any disorder in which the blood vessels of a subject(preferably a human adult, child or infant) become inflamed and receiveincreased blood flow, causing visible redness, intense heat and burningpain, usually in the upper or lower extremities (i.e. hands and/orfeet).

Erythromelalgia may be primary erythromelalgia (i.e. a disorder initself) or secondary erythromelalgia (i.e. a symptom of anothercondition). Primary erythromelalgia is an autosomal dominant disorderthought to be caused by a mutation in the Na_(v)1.7 sodium ion channelgene, SCN9A. Secondary erythromelalgia may result as a symptom ofanother disorder, such as bone marrow disorders (e.g. myeloproliferativedisorders), neuropathies, certain autoimmune connective tissue diseases,or result from the administration of certain drugs (e.g. bromocriptine,nifedipine, nicardipine), or mushroom or mercury poisoning.

In one embodiment, the disorder is selected from primary erythromelalgiaor secondary erythromelalgia. In a further embodiment, the disorder isprimary erythromelalgia. In an alternative embodiment, the disorder issecondary erythromelalgia.

The term “subject” as used herein, refers to an animal, preferably amammal, most preferably a human adult, child or infant, who has been theobject of treatment, observation or experiment.

It will be appreciated that references herein to “treatment” extend toprophylaxis, prevention of recurrence and suppression or amelioration ofsymptoms (whether mild, moderate or severe) as well as the treatment ofestablished conditions.

The term “therapeutically effective amount” as used herein, means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of one or more of thesymptoms of the disease or disorder being treated; and/or reduction ofthe severity of one or more of the symptoms of the disease or disorderbeing treated.

(5R)-5-(4-{[(2-Fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof may also beused in combination with other therapeutic agents. The invention thusprovides, in a further aspect, a combination comprising(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof togetherwith a further therapeutic agent for use in the treatment oferythromelalgia.

(5R)-5-(4-{[(2-Fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof may be usedin combination with other medicaments indicated to be useful in thetreatment or prophylaxis of pain (i.e. analgesics). Such therapeuticagents include for example COX-2 (cyclooxygenase-2) inhibitors, such ascelecoxib, deracoxib, rofecoxib, valdecoxib, parecoxib, COX-189 or2-(4-ethoxy-phenyl)-3-(4-methanesulfonyl-phenyl)-pyrazolo[1,5-b]pyridazine(WO 99/012930); 5-lipoxygenase inhibitors; NSAIDs (non-steroidalanti-inflammatory drugs) such as diclofenac, indomethacin, nabumetone oribuprofen; bisphosphonates, leukotriene receptor antagonists; DMARDs(disease modifying anti-rheumatic drugs) such as methotrexate; adenosineA1 receptor agonists; sodium channel blockers, such as lamotrigine; NMDA(N-methyl-D-aspartate) receptor modulators, such as glycine receptorantagonists or memantine; ligands for the α2S-subunit of voltage gatedcalcium channels, such as gabapentin, pregabalin and solzira; tricyclicantidepressants such as amitriptyline; neurone stabilising antiepilepticdrugs; cholinesterase inhibitors such as galantamine; mono-aminergicuptake inhibitors such as venlafaxine; opioid analgesics; localanaesthetics; 5HTI agonists, such as triptans, for example sumatriptan,naratriptan, zolmitriptan, eletriptan, frovatriptan, almotriptan orrizatriptan; nicotinic acetyl choline (nACh) receptor modulators;glutamate receptor modulators, for example modulators of the NR2Bsubtype; EP₄ receptor ligands; EP₂ receptor ligands; EP₃ receptorligands; EP4 agonists and EP2 agonists; EP4 antagonists; EP2 antagonistsand EP3 antagonists; cannabinoid receptor ligands; bradykinin receptorligands; vanilloid receptor or Transient Receptor Potential (TRP)ligands; and purinergic receptor ligands, including antagonists at P2X3,P2X2/3, P2X4, P2X7 or P2X4/7; KCNQ/Kv7 channel openers, such asretigabine; additional COX-2 inhibitors are disclosed in U.S. Pat. Nos.5,474,995, 5,633,272, 5,466,823, 6,310,099 and 6,291,523; and in WO96/25405, WO 97/38986, WO 98/03484, WO 97/14691, WO 99/12930, WO00/26216, WO 00/52008, WO 00/38311, WO 01/58881 and WO 02/18374.

In one embodiment, the present invention is directed to co-therapy,adjunctive therapy or combination therapy, comprising administration of(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof and one ormore analgesics (e.g. tramadol or amitriptyline), anticonvulsant drugs(e.g. gabapentin, neurontin or pregabalin (i.e. Lyrica)) orantidepressant drugs (e.g. duloxetine (i.e. Cymbalta) or venlafaxine).

In this embodiment, therapeutically effective amount shall mean thatamount of the combination of agents taken together so that the combinedeffect elicits the desired biological or medicinal response. Forexample, the therapeutically effective amount of co-therapy comprisingadministration of(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof and atleast one suitable analgesic, anticonvulsant or antidepressant drugswould be the amount of(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof and theamount of the suitable analgesic, anticonvulsant or antidepressant drugsthat when taken together or sequentially have a combined effect that istherapeutically effective. Further, it will be recognized by one skilledin the art that in the case of co-therapy with a therapeuticallyeffective amount, the amount of(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof and/or theamount of the suitable analgesic, anticonvulsant or antidepressant drugsindividually may or may not be therapeutically effective.

As used herein, the terms “co-therapy”, “adjunctive therapy” and“combination therapy” shall mean treatment of a subject in need thereofby administering one or more analgesic, anticonvulsant or antidepressantagent(s) and(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof, wherein(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof and theanalgesic, anticonvulsant or antidepressant agent(s) are administered byany suitable means, simultaneously, sequentially, separately or in asingle pharmaceutical formulation.

When administration is sequential, either the compound of the inventionor the second therapeutic agent may be administered first. Whenadministration is simultaneous, the combination may be administeredeither in the same or different pharmaceutical composition.

When combined in the same formulation it will be appreciated that thetwo compounds must be stable and compatible with each other and theother components of the formulation. When formulated separately they maybe provided in any convenient formulation, conveniently in such manneras are known for such compounds in the art.

Where (5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof and theanalgesic, anticonvulsant or antidepressant agent(s) are administered inseparate dosage forms, the number of dosages administered per day foreach compound may be the same or different.(5R)-5-(4-{[(2-Fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof and theanalgesic, anticonvulsant or antidepressant agent(s) may be administeredvia the same or different routes of administration. Examples of suitablemethods of administration include, but are not limited to, oral,intravenous (iv), intramuscular (im), subcutaneous (sc), intranasal,transdermal, and rectal. Compounds may also be administered directly tothe nervous system including, but not limited to, intracerebral,intraventricular, intracerebroventhcular, intrathecal, intracisternal,intraspinal and/or peri-spinal routes of administration by delivery viaintracranial or intravertebral needles and/or catheters with or withoutpump devices.(5R)-5-(4-{[(2-Fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof and theanalgesic, anticonvulsant or antidepressant agent(s) may be administeredaccording to simultaneous or alternating regimens, at the same ordifferent times during the course of the therapy, concurrently individed or single forms.

Advantageously,(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof may beadministered in a single daily dose, or the total daily dosage may beadministered in divided doses of two, three or four times daily.

The present invention provides methods of treating erythromelalgiacomprising administering to a subject in need thereof, a therapeuticallyeffective amount of(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof asdescribed herein.

(5R)-5-(4-{[(2-Fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof may beadministered as the raw chemical but the active ingredient is preferablypresented as a pharmaceutical composition.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombinations of the specified ingredients in the specified amounts.

Since the compounds described herein are intended for use inpharmaceutical compositions it will readily be understood that they areeach preferably provided in substantially pure form, for example atleast 60% pure, more suitably at least 75% pure and preferably at least85%, especially at least 98% pure (% are given on a weight for weightbasis). Impure preparations of the compounds may be used for preparingthe more pure forms used in the pharmaceutical compositions.

According to a further aspect of the invention, there is provided apharmaceutical composition comprising(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof for use inthe treatment of erythromelalgia.

In one embodiment, the pharmaceutical composition comprises one or morepharmaceutically acceptable carrier(s), diluent(s) and/or excipient(s).The carrier, diluent and/or excipient must be “acceptable” in the senseof being compatible with the other ingredients of the composition andnot deleterious to the recipient thereof.

Pharmaceutical compositions containing(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof as theactive ingredient can be prepared by intimately mixing the compound witha pharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. These procedures may involve mixing, granulatingand compressing or dissolving the ingredients as appropriate to thedesired preparation.

The compounds of the invention may be administered in conventionaldosage forms prepared by combining a compound of the invention withstandard pharmaceutical carriers or diluents according to conventionalprocedures well known in the art. These procedures may involve mixing,granulating and compressing or dissolving the ingredients as appropriateto the desired preparation.

The compounds or their pharmaceutically acceptable salts may beadministered by any convenient method, e.g. by oral, parenteral, buccal,sublingual, nasal, rectal or transdermal administration, and thepharmaceutical compositions adapted accordingly, for administration tomammals including humans.

The compounds or their pharmaceutically acceptable salts which areactive when given orally can be formulated as liquids or solids, e.g. assyrups, suspensions, emulsions, tablets, capsules or lozenges.

The topical formulations of the present invention may be presented as,for instance, ointments, creams or lotions, eye ointments and eye or eardrops, impregnated dressings and aerosols, and may contain appropriateconventional additives such as preservatives, solvents to assist drugpenetration and emollients in ointments and creams.

The formulations may also contain compatible conventional carriers, suchas cream or ointment bases and ethanol or oleyl alcohol for lotions.Such carriers may be present as from about 1% up to about 98% of theformulation. More usually they will form up to about 80% of theformulation.

A liquid formulation will generally consist of a suspension or solutionof the active ingredient in a suitable liquid carrier(s) e.g. an aqueoussolvent such as water, ethanol or glycerine, or a non-aqueous solvent,such as polyethylene glycol or an oil. The formulation may also containa suspending agent, preservative, flavouring and/or colouring agent.

Tablets and capsules for oral administration may be in unit dosepresentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatine, sorbitol,tragacanth, or polyvinylpyrrolidone; fillers, for example lactose,sugar, maize starch, calcium phosphate, sorbitol or glycine; tabletinglubricants, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants, for example potato starch; or acceptable wettingagents such as sodium lauryl sulphate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives, such as suspending agents, for example sorbitol,methyl cellulose, glucose syrup, gelatine, hydroxyethyl cellulose,carboxymethyl cellulose, aluminium stearate gel or hydrogenated ediblefats, emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non aqueous vehicles (which may include edible oils), forexample almond oil, oily esters such as glycerine, propylene glycol, orethyl alcohol; preservatives, for example methyl or propyl phydroxybenzoate or sorbic acid, and, if desired, conventional flavouringor colouring agents.

Typical parenteral compositions consist of a solution or suspension ofthe active ingredient in a sterile vehicle, water being preferred, orparenterally acceptable oil, e.g. polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, thesolution can be lyophilised and then reconstituted with a suitablesolvent just prior to administration. The compound, depending on thevehicle and concentration used, can be either suspended or dissolved inthe vehicle. In preparing solutions the compound can be dissolved inwater for injection and filter-sterilised before filling into a suitablevial or ampoule and sealing.

Advantageously, agents such as local anaesthetics, preservatives andbuffering agents can be dissolved in the vehicle. To enhance thestability, the composition can be frozen after filling into the vial andthe water removed under vacuum. The dry lyophilised powder is thensealed in the vial and an accompanying vial of water for injection maybe supplied to reconstitute the liquid prior to use. Parenteralsuspensions are prepared in substantially the same manner except thatthe compound is suspended in the vehicle instead of being dissolved andsterilisation cannot be accomplished by filtration. The compound can besterilised by exposure to ethylene oxide before suspending in thesterile vehicle. Advantageously, a surfactant or wetting agent isincluded in the composition to facilitate uniform distribution of thecompound.

Compositions for nasal administration may conveniently be formulated asaerosols, drops, gels and powders. Aerosol formulations typicallycomprise a solution or fine suspension of the active ingredient in apharmaceutically acceptable aqueous or non-aqueous solvent and areusually presented in single or multidose quantities in sterile form in asealed container which can take the form of a cartridge or refill foruse with an atomising device. Alternatively the sealed container may bea disposable dispensing device such as a single dose nasal inhaler or anaerosol dispenser fitted with a metering valve. Where the dosage formcomprises an aerosol dispenser, it will contain a propellant which canbe a compressed gas e.g. air, or an organic propellant such as afluoro-chloro-hydro-carbon or hydrofluorocarbon. Aerosol dosage formscan also take the form of pump-atomisers.

Compositions suitable for buccal or sublingual administration includetablets, lozenges and pastilles where the active ingredient isformulated with a carrier such as sugar and acacia, tragacanth, orgelatin and glycerin. Compositions for rectal administration areconveniently in the form of suppositories containing a conventionalsuppository base such as cocoa butter. Compositions suitable fortransdermal administration include ointments, gels and patches.

In one embodiment the composition is in unit dose form such as a tablet,capsule or ampoule.

The dose of the compound or a pharmaceutically acceptable salt thereof,used in the treatment of the abovementioned disorders or diseases willvary in the usual way with the particular disorder or disease beingtreated, the weight of the subject and other similar factors. However,as a general rule, suitable unit doses may contain from 0.1% to 100% byweight, for example from 10 to 60% by weight, of the active material,depending on the method of administration. The composition may containfrom 0% to 99% by weight, for example 40% to 90% by weight, of thecarrier, depending on the method of administration. The composition maycontain from 0.05 mg to 1000 mg, for example from 1.0 mg to 500 mg, ofthe active material, depending on the method of administration. Thecomposition may contain from 50 mg to 1000 mg, for example from 100 mgto 400 mg of the carrier, depending on the method of administration. Thedose of the compound used in the treatment of the aforementioneddisorders will vary in the usual way with the seriousness of thedisorders, the weight of the sufferer, and other similar factors.However, as a general guide suitable unit doses may be in the range of50 mg to 1500 mg per day, for example 120 mg to 1000 mg per day. Suchtherapy may extend for a number of weeks or months.

It will be recognised by one of skill in the art that the optimalquantity and spacing of individual dosages of(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof will bedetermined by the nature and extent of the condition being treated, theform, route and site of administration, and the particular mammal beingtreated, and that such optimums can be determined by conventionaltechniques. In addition, factors associated with the particular patientbeing treated, including patient age, weight, diet and time ofadministration, will result in the need to adjust dosages. It will alsobe appreciated by one of skill in the art that the optimal course oftreatment, i.e., the number of doses of a compound of the inventiongiven per day for a defined number of days, can be ascertained by thoseskilled in the art using conventional course of treatment determinationtests.

Diagnosis

According to a further aspect of the invention, there is provided amethod of diagnosing erythromelalgia in a human subject wherein saidmethod comprises detecting the presence of one or more geneticvariations within the CACNA1A gene and/or the CACNA1B gene of saidsubject.

References herein to “CACNA1A gene” refer to the calcium channel,voltage-dependent, P/Q type, alpha 1A subunit gene (also known asCav2.1). The CACNA1A gene belongs to a family of genes that code for thepore forming alpha subunits of calcium channels. These channels, whichtransport positively charged calcium atoms (calcium ions) across cellmembranes, play a key role in a cell's ability to generate and transmitelectrical signals. Calcium ions are involved in many different cellularfunctions, including cell-to-cell communication, the tensing of musclefibers (muscle contraction), and the regulation of certain genes.

The CACNA1A gene provides instructions for making one part (the alpha-1subunit) of a calcium channel called Cav2.1. This subunit forms the hole(pore) through which calcium ions can flow. Cav2.1 channels play anessential role in communication between nerve cells (neurons) in thebrain and spinal cord. These channels help control the release ofneurotransmitters, which are chemicals that relay signals from oneneuron to another. It is believed that Cav2.1 channels are also involvedin the survival of neurons and the ability of these cells to change andadapt over time (plasticity).

Near one end of the CACNA1A gene, a segment of three DNA building blocks(nucleotides) is repeated multiple times. This sequence, which iswritten as CAG, is called a triplet or trinucleotide repeat. In mostpeople, the number of CAG repeats in this gene ranges from 4 to 18.

The CACNA1A gene is located on the short (p) arm of chromosome 19 atposition 13 (19p13). More precisely, the molecular location of theCACNA1A gene is on chromosome 19: base pairs 13,206,441 to 13,506,459.

CACNA1A has been previously linked with episodic ataxia, familialhemiplegic migraine, spinocerebellar ataxia type 6 and sporadichemiplegic migraine.

References herein to “CACNA1B gene” refer to the calcium channel,voltage-dependent, N type, alpha 1B subunit gene (also known as Cav2.2).The protein encoded by this gene is the pore-forming subunit of anN-type voltage-dependent calcium channel, which controlsneurotransmitter release from neurons. The encoded protein forms acomplex with alpha-2, beta, and delta subunits to form the high-voltageactivated channel. This channel is sensitive to omega-conotoxin-GVIA andomega-agatoxin-IIIA but insensitive to dihydropyridines. Two transcriptvariants encoding different isoforms have been found for this gene.

The CACNA1B gene is located on the long (q) arm of chromosome 9 atposition 34 (9q34). More precisely, the molecular location of theCACNA1B gene is on chromosome 9: base pairs 140,772,241 to 141,019,076.

Diseases associated with CACNA1B include krukenberg carcinoma, andmorvan's fibrillary chorea, and among its related super-pathways aresynaptic vesicle pathway and circadian entrainment.

References herein to “genetic variation” refer to a difference orvariation between the genetic code or sequence of a human subject whencompared with a wild-type or native genetic code or sequence. It will beappreciated that the genetic variations include any variation in thewild type or native genetic code of the genome from said human subjectunder analysis. Examples of such genetic variations include: mutations(e.g. point mutations), insertions, substitutions, deletions, frameshifts, single nucleotide polymorphisms (SNPs), haplotypes, chromosomeabnormalities, copy number variation (CNV), epigenetics and DNAinversions. In one embodiment, the genetic variations are selected fromsubstitutions, insertions, deletions and frame shifts.

References herein to the term “single-nucleotide polymorphism (SNP)” isintended to refer to DNA sequence variation occurring when a singlenucleotide in the genome (or other shared sequence) differs betweenmembers of a species or between paired chromosomes in an individual.

References herein to the term “haplotype” is intended to refer to a setof genetic markers that are inherited together as a consequence of theirchromosomal co-localization. Haplotype may refer to as few as twogenetic variants or to an entire chromosome depending on the number ofrecombination events that have occurred between a given set of variants.

In one embodiment, the genetic variations are detected within theCACNA1A gene. In a further embodiment, the genetic variations are withinthe CACNA1A gene and are between 13,300,000 bp and 13,450,000 bp onchromosome 19, such as between 13,318,000 bp and 13,412,000 bp onchromosome 19.

The methods of the invention may be used to detect genetic variationsusing a biological sample obtained from the human subject. Thus, in oneembodiment, the method initially comprises the step of obtaining abiological sample from the human subject.

The nucleic acid may be isolated from the sample according to anymethods well known to those of skill in the art. Examples include tissuesamples or any cell-containing or a cellular biomaterial (i.e. a bodilyfluid or hair sample). Biological samples may be obtained by standardprocedures and may be used immediately or stored, under conditionsappropriate for the type of biological sample, for later use.

Methods of obtaining biological samples are well known to those of skillin the art and include, but are not limited to, aspirations, tissuesections, drawing of blood or other fluids, surgical or needle biopsies,and the like. Examples of suitable biological samples include: wholeblood, blood serum, plasma, urine, saliva, or other bodily fluid (stool,tear fluid, synovial fluid, sputum), hair, cerebrospinal fluid (CSF), oran extract or purification therefrom, or dilution thereof. Biologicalsamples also include tissue homogenates, tissue sections and biopsyspecimens from a live subject. The samples can be prepared, for examplewhere appropriate diluted or concentrated, and stored in the usualmanner. In one embodiment, the biological sample is blood or serum. In afurther embodiment, the biological sample is blood.

If necessary, the sample may be collected or concentrated bycentrifugation and the like. The cells of the sample may be subjected tolysis, such as by treatments with enzymes, heat, surfactants,ultrasonication, or a combination thereof. The lysis treatment isperformed in order to obtain a sufficient amount of nucleic acid derivedfrom the cells of the human subject to detect using polymerase chainreaction.

Methods of plasma and serum preparation are well known in the art.Either “fresh” blood plasma or serum, or frozen (stored) andsubsequently thawed plasma or serum may be used. Frozen (stored) plasmaor serum should optimally be maintained at storage conditions of −20 to−70° C. until thawed and used. “Fresh” plasma or serum should berefrigerated or maintained on ice until used, with nucleic acid (e.g.,RNA, DNA or total nucleic acid) extraction being performed as soon aspossible. Exemplary methods are described below.

Blood can be drawn by standard methods into a collection tube, typicallysiliconized glass, either without anticoagulant for preparation ofserum, or with EDTA, sodium citrate, heparin, or similar anticoagulantsfor preparation of plasma. If preparing plasma or serum for storage,although not an absolute requirement, plasma or serum is firstfractionated from whole blood prior to being frozen. This reduces theburden of extraneous intracellular RNA released from lysis of frozen andthawed cells which might reduce the sensitivity of the amplificationassay or interfere with the amplification assay through release ofinhibitors to PCR such as porphyrins and hematin. “Fresh” plasma orserum may be fractionated from whole blood by centrifugation, usinggentle centrifugation at 300-800 times gravity for five to ten minutes,or fractionated by other standard methods. High centrifugation ratescapable of fractionating out apoptotic bodies should be avoided.

It will be appreciated that the step of detecting the one or moregenetic variations within the regions defined herein will comprise anysuitable technique for genetic analysis.

The volume of plasma or serum used in the extraction may vary, butvolumes of 1 to 100 ml of plasma or serum are usually sufficient.

Various methods of extraction are suitable for isolating the nucleicacid. Suitable methods include phenol and chloroform extraction. SeeManiatis et al., Molecular Cloning, A Laboratory Manual, 2d, Cold SpringHarbor Laboratory Press, page 16.54 (1989).

Numerous commercial kits also yield suitable DNA and RNA including, butnot limited to, QIAamp™ mini blood kit, Agencourt Genfind™, Roche Cobas®Roche MagNA Pure® or phenol: chloroform extraction using Eppendorf PhaseLock Gels®, and the NucliSens extraction kit (Biomerieux, Marcyl'Etoile, France). In other methods, mRNA may be extracted fromblood/bone marrow samples using MagNA Pure LC mRNA HS kit and Mag NAPure LC Instrument (Roche Diagnostics Corporation, Roche AppliedScience, Indianapolis, Ind.).

Nucleic acid extracted from tissues, cells, plasma, serum or hair can beamplified using nucleic acid amplification techniques well known in theart. Many of these amplification methods can also be used to detect thepresence of genetic variations simply by designing oligonucleotideprimers or probes to interact with or hybridize to a particular targetsequence in a specific manner. By way of example, but not by way oflimitation, these techniques can include the polymerase chain reaction(PCR), reverse transcriptase polymerase chain reaction (RT-PCR), nestedPCR, ligase chain reaction. See Abravaya, K., et al, Nucleic AcidsResearch, 23:675-682, (1995), branched DNA signal amplification, Urdea,M. S., et al, AIDS, 7 (suppl 2):S11-S14, (1993), amplifiable RNAreporters, Q-beta replication, transcription-based amplification,boomerang DNA amplification, strand displacement activation, cyclingprobe technology, isothermal nucleic acid sequence based amplification(NASBA). See Kievits, T. et al, J Virological Methods, 35:273-286,(1991), Invader Technology, or other sequence replication assays orsignal amplification assays. These methods of amplification eachdescribed briefly below and are well-known in the art.

Some methods employ reverse transcription of RNA to cDNA. Variousreverse transcriptases may be used, including, but not limited to, MMLVRT, RNase H mutants of MMLV RT such as Superscript and Superscript II(Life Technologies, GIBCO BRL, Gaithersburg, Md.), AMV RT, andthermostable reverse transcriptase from Thermus Thermophilus. Forexample, one method, but not the only method, which may be used toconvert RNA extracted from plasma or serum to cDNA is the protocoladapted from the Superscript II Preamplification system (LifeTechnologies, GIBCO BRL, Gaithersburg, Md.; catalog no. 18089-011), asdescribed by Rashtchian, A., PCR Methods Applic, 4:S83-S91, (1994).

A variety of amplification enzymes are well known in the art andinclude, for example, DNA polymerase, RNA polymerase, reversetranscriptase, Q-beta replicase, thermostable DNA and RNA polymerases.Because these and other amplification reactions are catalyzed byenzymes, in a single step assay the nucleic acid releasing reagents andthe detection reagents should not be potential inhibitors ofamplification enzymes if the ultimate detection is to be amplificationbased. Amplification methods suitable for use with the present methodsinclude, for example, strand displacement amplification, rolling circleamplification, primer extension preamplification, or degenerateoligonucleotide PCR (DOP).

In one embodiment, PCR is used to amplify a target or marker sequence ofinterest. The person skilled in the art is capable of designing andpreparing primers that are appropriate for amplifying a target sequence.The length of the amplification primers depends on several factorsincluding the nucleotide sequence identity and the temperature at whichthese nucleic acids are hybridized or used during in vitro nucleic acidamplification. The considerations necessary to determine a preferredlength for an amplification primer of a particular sequence identity arewell-known to the person skilled in the art. For example, the length ofa short nucleic acid or oligonucleotide can relate to its hybridizationspecificity or selectivity.

For analyzing SNPs and other variant nucleic acids, it may beappropriate to use oligonucleotides specific for alternative alleles.Such oligonucleotides which detect single nucleotide variations intarget sequences may be referred to by such terms as “allele-specificprobes”, or “allele-specific primers”. The design and use ofallele-specific probes for analyzing polymorphisms is described in,e.g., Mutation Detection A Practical Approach, ed. Cotton et al. OxfordUniversity Press, 1998; Saiki et al, Nature, 324: 163-166 (1986);Dattagupta, EP235,726; and Saiki, WO 89/11548. In one embodiment, aprobe or primer may be designed to hybridize to a segment of target DNAsuch that the SNP aligns with either the 5′ most end or the 3′ most endof the probe or primer.

In some embodiments, the amplification may include a labeled primer,thereby allowing detection of the amplification product of that primer.In particular embodiments, the amplification may include a multiplicityof labeled primers; typically, such primers are distinguishably labeled,allowing the simultaneous detection of multiple amplification products.

In one type of PCR-based assay, an allele-specific primer hybridizes toa region on a target nucleic acid molecule that overlaps a SNP positionand only primes amplification of an allelic form to which the primerexhibits perfect complementarity (Gibbs, 1989, Nucleic Acid Res.,17:2427-2448). Typically, the primer's 3′-most nucleotide is alignedwith and complementary to the SNP position of the target nucleic acidmolecule. This primer is used in conjunction with a second primer thathybridizes at a distal site. Amplification proceeds from the twoprimers, producing a detectable product that indicates which allelicform is present in the test sample. A control is usually performed witha second pair of primers, one of which shows a single base mismatch atthe polymorphic site and the other of which exhibits perfectcomplementarity to a distal site. The single-base mismatch preventsamplification or substantially reduces amplification efficiency, so thateither no detectable product is formed or it is formed in lower amountsor at a slower pace. The method generally works most effectively whenthe mismatch is at the 3′-most position of the oligonucleotide (i.e.,the 3′-most position of the oligonucleotide aligns with the target SNPposition) because this position is most destabilizing to elongation fromthe primer (see, e.g., WO 93/22456).

In a specific embodiment, a primer contains a sequence substantiallycomplementary to a segment of a target SNP-containing nucleic acidmolecule except that the primer has a mismatched nucleotide in one ofthe three nucleotide positions at the 3′-most end of the primer, suchthat the mismatched nucleotide does not base pair with a particularallele at the SNP site. In one embodiment, the mismatched nucleotide inthe primer is the second from the last nucleotide at the 3′-mostposition of the primer. In another embodiment, the mismatched nucleotidein the primer is the last nucleotide at the 3′-most position of theprimer.

In one embodiment, primer or probe is labeled with a fluorogenicreporter dye that emits a detectable signal. While a suitable reporterdye is a fluorescent dye, any reporter dye that can be attached to adetection reagent such as an oligonucleotide probe or primer is suitablefor use in the invention. Such dyes include, but are not limited to,Acridine, AMCA, BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Dabcyl, Edans,Eosin, Erythrosin, Fluorescein, 6-Fam, Tet, Joe, Hex, Oregon Green,Rhodamine, Rhodol Green, Tamra, Rox, and Texas Red.

It will be appreciated that the invention extends to reagents that donot contain (or that are complementary to) a SNP nucleotide identifiedherein but that are used to assay one or more SNPs disclosed herein. Forexample, primers that flank, but do not hybridize directly to a targetSNP position provided herein are useful in primer extension reactions inwhich the primers hybridize to a region adjacent to the target SNPposition (i.e., within one or more nucleotides from the target SNPsite). During the primer extension reaction, a primer is typically notable to extend past a target SNP site if a particular nucleotide(allele) is present at that target SNP site, and the primer extensionproduct can readily be detected in order to determine which SNP alleleis present at the target SNP site. For example, particular ddNTPs aretypically used in the primer extension reaction to terminate primerextension once a ddNTP is incorporated into the extension product. Thus,reagents that bind to a nucleic acid molecule in a region adjacent to aSNP site, even though the bound sequences do not necessarily include theSNP site itself, are also encompassed by the invention.

Variant nucleic acids may be amplified prior to detection or may bedetected directly during an amplification step (i.e., “real-time”methods). In some embodiments, the target sequence is amplified and theresulting amplicon is detected by electrophoresis. In some embodiments,the specific mutation or variant is detected by sequencing the amplifiednucleic acid. In some embodiments, the target sequence is amplifiedusing a labeled primer such that the resulting amplicon is detectablylabeled. In some embodiments, the primer is fluorescently labeled.

In one embodiment, detection of a variant nucleic acid, such as a SNP,is performed using the TaqMan® assay, which is also known as the 5′nuclease assay (U.S. Pat. Nos. 5,210,015 and 5,538,848) or MolecularBeacon probe (U.S. Pat. Nos. 5,118,801 and 5,312,728), or other stemlessor linear beacon probe (Livak et al, 1995, PCR Method AppL, 4:357-362;Tyagi et al, 1996, Nature Biotechnology, 14:303-308; Nazarenko et al,1997, Nucl. Acids Res., 25:2516-2521; U.S. Pat. Nos. 5,866,336 and6,117,635). The TaqMan® assay detects the accumulation of a specificamplified product during PCR. The TaqMan® assay utilizes anoligonucleotide probe labeled with a fluorescent reporter dye and aquencher dye. The reporter dye is excited by irradiation at anappropriate wavelength, it transfers energy to the quencher dye in thesame probe via a process called fluorescence resonance energy transfer(FRET). When attached to the probe, the excited reporter dye does notemit a signal. The proximity of the quencher dye to the reporter dye inthe intact probe maintains a reduced fluorescence for the reporter. Thereporter dye and quencher dye may be at the 5′ most and the 3′ mostends, respectively or vice versa. Alternatively, the reporter dye may beat the 5′ or 3′ most end while the quencher dye is attached to aninternal nucleotide, or vice versa. In yet another embodiment, both thereporter and the quencher may be attached to internal nucleotides at adistance from each other such that fluorescence of the reporter isreduced.

During PCR, the 5′ nuclease activity of DNA polymerase cleaves theprobe, thereby separating the reporter dye and the quencher dye andresulting in increased fluorescence of the reporter. Accumulation of PCRproduct is detected directly by monitoring the increase in fluorescenceof the reporter dye. The DNA polymerase cleaves the probe between thereporter dye and the quencher dye only if the probe hybridizes to thetarget SNP-containing template which is amplified during PCR, and theprobe is designed to hybridize to the target SNP site only if aparticular SNP allele is present.

TaqMan® primer and probe sequences can readily be determined using thevariant and associated nucleic acid sequence information providedherein. A number of computer programs, such as Primer Express (AppliedBiosystems, Foster City, Calif.), can be used to rapidly obtain optimalprimer/probe sets. It will be apparent to one of skill in the art thatsuch primers and probes for detecting the genetic variations of theinvention are useful in diagnosis of erythromelalgia, and can be readilyincorporated into a kit format. The invention also includesmodifications of the TaqMan® assay well known in the art such as the useof Molecular Beacon probes (U.S. Pat. Nos. 5,118,801 and 5,312,728) andother variant formats (U.S. Pat. Nos. 5,866,336 and 6,117,635).

Other methods of probe hybridization detected in real time can be usedfor detecting amplification of a target or marker sequence flanking atandem repeat region. For example, the commercially available MGBEclipse™ probes (Epoch Biosciences), which do not rely on a probedegradation can be used. MGB Eclipse™ probes work by ahybridization-triggered fluorescence mechanism. MGB Eclipse™ probes havethe Eclipse™ Dark Quencher and the MGB positioned at the 5′-end of theprobe. The fluorophore is located on the 3′-end of the probe. When theprobe is in solution and not hybridized, the three dimensionalconformation brings the quencher into close proximity of thefluorophore, and the fluorescence is quenched. However, when the probeanneals to a target or marker sequence, the probe is unfolded, thequencher is moved from the fluorophore, and the resultant fluorescencecan be detected.

Oligonucleotide probes can be designed which are between about 10 andabout 100 nucleotides in length and hybridize to the amplified region.Oligonucleotides probes are preferably 12 to 70 nucleotides; morepreferably 15-60 nucleotides in length; and most preferably 15-25nucleotides in length. The probe may be labeled. Amplified fragments maybe detected using standard gel electrophoresis methods. For example, inpreferred embodiments, amplified fractions are separated on an agarosegel and stained with ethidium bromide by methods known in the art todetect amplified fragments.

Another suitable detection methodology involves the design and use ofbipartite primer/probe combinations such as Scorpion™ probes. Theseprobes perform sequence-specific priming and PCR product detection isachieved using a single molecule. Scorpion™ probes comprise a 3′ primerwith a 5′ extended probe tail comprising a hairpin structure whichpossesses a fluorophore/quencher pair. The probe tail is “protected”from replication in the 5′ to 3′ direction by the inclusion ofhexethlyene glycol (HEG) which blocks the polymerase from replicatingthe probe. The fluorophore is attached to the 5′ end and is quenched bya moiety coupled to the 3′ end. After extension of the Scorpion™ primer,the specific probe sequence is able to bind to its complement within theextended amplicon thus opening up the hairpin loop. This prevents thefluorescence from being quenched and a signal is observed. A specifictarget is amplified by the reverse primer and the primer portion of theScorpion™, resulting in an extension product. A fluorescent signal isgenerated due to the separation of the fluorophore from the quencherresulting from the binding of the probe element of the Scorpion™ to theextension product. Such probes are described in Whitcombe et al, NatureBiotech 17: 804-807 (1999). Determining Prognosis According to a furtheraspect of the invention there is provided a kit for diagnosingerythromelalgia which comprises instructions to use said kit inaccordance with the methods defined herein.

The kits may be prepared for practicing the methods described herein.Typically, the kits include at least one component or a packagedcombination of components useful for practicing the method of theinvention. The kits may include some or all of the components necessaryto practice method of the invention. Typically, the kits include atleast one probe specific for the one or more regions defined herein inat least one container. These components may include, inter alia,nucleic acid probes, nucleic acid primers for amplification of the oneor more regions defined herein, buffers, instructions for use, and thelike.

It will be appreciated that once an individual has been identified aslikely to suffer from or suffering from erythromelalgia, said individualwill then be prescribed suitable treatment, such as(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof.

Thus, according to a further aspect of the invention, there is provideda method of treating erythromelalgia in a human subject wherein saidmethod comprises:

-   -   (a) detecting the presence of one or more genetic variations        within the CACNA1A gene and/or the CACNA1B gene of said subject;        and    -   (b) administering        (5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or        a pharmaceutically acceptable salt, solvate or prodrug thereof        to said patient identified as having said one or more genetic        variations.

As discussed hereinbefore, current treatment for erythromelalgia isassociated with poor tolerability and results in sub-optimal paincontrol. There is therefore a great need to identify individuals mostlikely to respond to such therapy.

Thus, according to a further aspect of the invention, there is provideda method of predicting whether an erythromelalgia patient will respondto treatment with(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof, whereinsaid method comprises the steps of:

-   -   (a) obtaining a biological sample from a patient; and    -   (b) detecting the presence of one or more genetic variations        within the CACNA1A gene and/or the CACNA1B gene of said subject;        such that the presence of said one or more genetic variations is        indicative that an erythromelalgia patient will respond to        treatment with        (5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or        a pharmaceutically acceptable salt, solvate or prodrug thereof.

It will be appreciated that once a “responder” has been identified, thepatient will then suitably be administered with(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof.

Thus, in one embodiment, the method additionally comprises:

-   -   (c) administering        (5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or        a pharmaceutically acceptable salt, solvate or prodrug thereof        to said erythromelalgia patient identified as having said one or        more genetic variations.

According to a further aspect of the invention, there is provided(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof for use inthe treatment of erythromelalgia in a patient, characterised in thatsaid patient has been selected for having one or more genetic variationswithin the CACNA1A gene and/or the CACNA1B gene.

According to a further aspect of the invention, there is provided amethod of treating erythromelalgia in a patient wherein said methodcomprises the steps of selecting a patient having one or more geneticvariations within the CACNA1A gene and/or the CACNA1B gene followed byadministering a therapeutically effective amount of(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide or apharmaceutically acceptable salt, solvate or prodrug thereof to saidpatient.

Throughout the specification and claims which follow, unless the contextrequires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’ will be understood to imply the inclusionof a stated integer or step or group of integers but not to theexclusion of any other integer or step or group of integers or steps.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

EXAMPLES

The invention is illustrated by the Examples described below:

Example 1: (5R)-5-(4-{[(2-Fluorophenyl)methyl]oxy}phenyl)-L-prolinamide(E1)

The compound of Example 1 may be prepared as described in Example 1,Procedures 1 and 2 of WO 2007/042239.

Example 2: (5R)-5-(4-{[(2-Fluorophenyl)methyl]oxy}phenyl)-L-prolinamideHydrochloride (E2)

The compound of Example 2 may be prepared as described in Example 2,Procedures 1 to 5 of WO 2007/042239.

Example 3: (5R)-5-(4-{[(2-Fluorophenyl)methyl]oxy}phenyl)-L-prolinamideMethanesulfonate (E3)

The compound of Example 3 may be prepared as described in Example 6 ofWO 2007/042239.

The following study illustrates the invention:

Whole cell patch-clamp electrophysiology was used to investigate theeffects of the compound of Example 2 on human recombinant WT (TTX-Rversion, Y362S) and 4 mutated Navy 1.7 channels from erythromelalgiapatients (V400M, I234T, F1449V and S241T), stably expressed in HEK293cells. Cell reagents were kindly provided by Prof. Stephen Waxman's lab,Yale.

Low frequency (0.1 Hz) stimulation (20 ms test pulse to 0 mV) from twodifferent holding potentials (Vhold) was used to assess thevoltage-dependent block by Example 2 and carbamazepine (CBZ). Firstly,Vhold=−90 mV, where most of the channels are in the resting state andsecondly Vhold=−60 mV (for V400M and 1234T) or Vhold=−70 mV (for F1449Vand S241T), which are a physiologically relevant neuronal restingpotential. The following solutions were used: External (mM): 140NaCl,3KCl, 1MgCl₂, 1CaCl₂, 10 Glucose and 20 HEPES, pH7.3, 320 mOsm; Internal(mM): 140CsF, 10 NaCl, 10 HEPES, 1EGTA, pH7.3, 310 mOsm. A Hill fit[Y=(V_(max)*X^(nH))/(IC50^(nH)+X^(nH))] on the data was used todetermine the IC₅₀ and the Hill Coefficient (nH).

Example 2 blocked the WT, V400M, 1234T, F1449V and S241T currents in avoltage-dependent fashion (see FIG. 1). pIC₅₀ (the negative log of theIC₅₀ concentration) and nH values are summarised in Table 1 and 2 below:

TABLE 1 WT pIC₅₀ V400M pIC₅₀ I234T pIC₅₀ (nH) (nH) (nH) Example 2 (Vhold= −90 mV) 4.5 (1) 4.3 (1) 4.1 (1) Example 2 (Vhold = −60 mV) 6 (0.6) 6(0.3) 6 (0.3) CBZ (Vhold = −90 mV) 3 (1) 3.2 (1) 3.3 (0.8) CBZ (Vhold =−60 mV) 4.2 (0.7) 4.2 (0.6) 3.9 (0.6)

TABLE 2 WT pIC₅₀ F1449V pIC₅₀ S241T pIC₅₀ (nH) (nH) (nH) Example 2(Vhold = −90 mV) 4.5 (1) 4.4 (0.9) 5.1 (0.8) Example 2 (Vhold = −70 mV)5.3 (0.7) 5.7 (0.4) 6.4 (0.3)

Example 2 has demonstrated efficacy in trigeminal neuralgia at 150 mgthree times a 5 day, the corresponding free (non protein bound)concentrations are 0.11 and 0.21 μM for Cmin and Cmax respectively. Usedat 200 mg four times a day, the range of Cmax free concentrations forCBZ is 4-10 μM. When the free clinical concentration of Example 2 andCBZ are reported on the respective concentration response plots, thelevel of inhibition of the channel in this particular assay can bemeasured (see Table 3 and 4):

TABLE 3 Vhold = −60 mV WT V400M I234T CBZ 13-21 14-22 12-19 Example 220-28 38-42 37-42

TABLE 4 (Vhold = −70 mV) WT F1449V S241T Example 2 5-9 21-28 44-49

At free clinical therapeutic concentration, Example 2 is more potentthan CBZ in the mutant channels compared to wild type, suggesting thatExample 2 differentiates from CBZ and will have specific utility for thetreatment of EM patients.

1-14. (canceled)
 15. A method of treating erythromelalgia, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of (5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide,or a pharmaceutically acceptable salt thereof.
 16. The method accordingto claim 15, wherein the(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide orpharmaceutically acceptable salt thereof is(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamidehydrochloride.
 17. The method according to claim 15, wherein the subjectis a human.
 18. The method according to claim 15, wherein theerythromelalgia is primary erythromelalgia.
 19. The method according toclaim 15, wherein the erythromelalgia is secondary erythromelalgia. 20.A method of treating erythromelalgia, comprising administering to asubject in need thereof a pharmaceutical composition comprising(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide, or apharmaceutically acceptable salt thereof.
 21. The method according toclaim 20, wherein the pharmaceutical composition further comprises oneor more pharmaceutically acceptable carriers, diluents, or excipients,or a combination thereof.
 22. The method according to claim 20, whereinthe (5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamide orpharmaceutically acceptable salt thereof is(5R)-5-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-L-prolinamidehydrochloride.
 23. The method according to claim 20, wherein theerythromelalgia is primary erythromelalgia.
 24. The method according toclaim 20, wherein the erythromelalgia is secondary erythromelalgia.